Hepatic stellate cells (HSCs) are the primary cell type responsible for hepatic fibrosis, the final common pathway leading to cirrhosis and liver failure for nearly every cause of chronic liver disease. Activation of HSCs in response to injury represents the key step in hepatic fibrogenesis, and is characterized by a phenotypic change from a non-fibrogenic, quiescent HSC to a fibrogenic myofibroblast that secretes extracellular matrix (ECM) proteins responsible for the fibrotic scar. There are currently no treatments available that are directed at the common endpoint of fibrosis. We have developed and performed a small molecule screen to identify compounds that revert fibrotic HSC myofibroblasts to the quiescent phenotype as an approach to discover molecular pathways that can inhibit hepatic fibrosis. We have successfully screened 1600 bioactive compounds and have identified 30 small molecules (1.9% hit rate) that induce HSC quiescence. Among the hits, we identified five tricyclic antidepressants (TCAs), and validated this class effect by measuring accumulation of fat droplets and repression of ACTA2 expression, both of which are characteristic of quiescent HSCs. We hypothesize that TCAs induce quiescence and inhibit fibrosis through the serotonin, acetylcholine, or sphingomyelinase pathways. We have defined the key components of each pathway that are expressed in human HSCs, and in Aim 1, we will test our hypothesis by performing loss of expression analysis with short hairpin (sh) RNAs and loss of function analysis with small molecule inhibitors to define the individual proteins involved in inducing quiescence. In addition, we will use genome-wide expression analysis to identify the dominant pathways affected by TCAs and to define the global effect of TCAs on expression of ECM factors. In Aim 2, we will quantify the effect of TCAs on liver fibrosis in the carbon tetrachloride (CCl4)-induced mouse model of fibrosis. The long-term goal of this work is to enhance our understanding of hepatic fibrogenesis and to develop therapies to prevent liver failure in patients with chronic liver disease. We anticipate that these studies will identify the molecular mechanism by which TCAs induce HSC quiescence and reduce liver fibrosis and lay the groundwork to develop new therapies to prevent the development of liver failure.